Novel, water-soluble porphyrin platinum compounds with high tumor selectivity and their use for the treatment of benign and malignant tumor diseases

ABSTRACT

The invention relates to novel, water-soluble porphyrin platinum compounds of the tetraarylporphyrin platinum derivatives type or of the hematoporphyrin platinum derivatives type with high tumor selectivity and their use for the treatment of benign and malignant tumor diseases. In particular, the compounds are suitable for photodynamic anti-tumor therapy.

INTRODUCTION

[0001] The invention relates to novel, water-soluble porphyrin platinumcompounds with high tumor selectivity and their use for the treatment ofbenign and malignant tumor diseases. In particular, the inventivecompounds are suitable for photodynamic anti-tumor therapy in man andmammals.

PRIOR ART

[0002] Platinum(II) complexes with porphyrin ligands and theirapplication as potent cytostatic and phototoxic antitumor agents havealready been described in the following publications.

[0003] W. M. Sharman, C. M. Allen and J. E. van Lier, DDT 4, (11)507-517 (1999). Photodynamic therapeutics: basic principles and clinicalapplications

[0004] T. Okunaka and H. Kato, Rev. Contemp. Pharmacother., 10, 59-68(1999). Potential Applications of Photodynamic Therapy.

[0005] H. Brunner, H. Obermeier and R.-M. Szeimies, Chem. Ber., 1995,128, 173-181. Platinum(II) complexes with porphyrin ligands: synthesisand synergism during photodynamic therapy.

[0006] H. Brunner, K.-H. Schellerer and B. Treittinger, Inorg. Chim.Acta 1997, 264, 67-69. Synthesis and in vitro testing of hematoporphyrintype ligands in platinum(II) complexes as potent cytostatic andphototoxic antitumor agents.

DESCRIPTION OF THE INVENTION

[0007] In the invention, novel porphyrin platinum derivatives aredescribed, which have cytotoxic properties. Surprisingly, the compoundshave good water solubility and a high selectivity. The compounds can beused for the treatment of cancer and, in particular, for thephotodynamic treatment of tumors.

[0008] The general formulas of the claimed compounds of thetetraarylporphyrin platinum derivatives type are:

[0009] The general formulas of the claimed compounds of thehematoporphyrin platinum derivatives type are:

[0010] If the inventive compounds have at least one center of asymmetry,they can be in the form of their racemates, their pure enantiomersand/or their diastereoisomers or in the form of mixtures of theseenantiomers or diastereoisomers.

[0011] The inventive compounds exhibit cytotoxic activity in selectedtumor cell lines. The antitumor activity is intensified by irradiatingwith electromagnetic radiation having a wavelength of 600 to 730 nm. Theinvention accordingly relates to the chemical combination of thecytotoxic principle of the platinum compounds of the cis platinum typewith a photodynamically active molecule of the porphyrin derivativetype, in such a manner, that compounds of good water solubility and highselectivity are obtained.

[0012] The inventive compounds can be administered intraaterially,intracerebrally, intramuscularly, intraperitoneally, intrathecally,intravenously, orally, parenterally, intranasally, rectally,subcutaneously and/or topically in the form of tablets, film-coatedtablets, capsules, coated tablets, powders, granulates, drops, syrups,ointments, powders for inhalation, infusion solutions, drinkingsolutions or in some other suitable form.

[0013] The medicaments comprise one or more compounds in addition tocustomary physiologically tolerable carriers and/or diluents orauxiliaries.

[0014] The process for the production of the medicament is characerizedin that one or more compounds are processed to give pharmaceuticalpreparations or brought into a therapeutically administrable form usingcustomary pharmaceutical carriers and/or diluents or other auxiliaries.

[0015] The synthesis of the inventive compounds is described.

[0016] Tetraarylporphyrin Platinum Derivatives

[0017] Synthesis of the substituted benzaldehydes. For the reaction with4-hydroxy-benzaldehyde the respective oligo- and polyethyleneglycolmonomethylethers had to be activated at their alcohol terminus withtosyl chloride according to a literature procedure. The etherificationwas performed by refluxing the tosylated alcohols and4-hydroxybenzaldehyde together with K₂CO₃ in DMF. The substitutedbenzaldehydes were separated by filtration and purified by columnchromatography.

[0018] For platinum coordination to the tetraarylporphyrins to besynthesized it is necessary to introduce two adjacent carboxylic acidgroups in one of the substituted benzaldehydes. Therefore,4-hydroxybenzaldehyde was etherified with diethyl bromomalonate underalkaline conditions. The diethyl 2-(4-formylphenoxy)malonate was usedtogether with the substituted benzaldehydes for the synthesis ofasymmetric tetraarylporphyrins.

[0019] Synthesis of the porphyrin ligands. The synthesis of theasymmetric tetraarylporphyrins was performed using the Lindsey method.Pyrrol and the respective benzaldehydes were reacted under Lewis acidcatalysis to porphyrinogens, which were oxidized with p-chloranil to thecorresponding porphyrins. The tetraarylporphyrin esters were purified byseveral column chromatographies. The carboxylic acids, which wererequired for coordination to the platinum(II) fragments, were preparedby hydrolysis of the esters with a mixture of CHCl₃ and 20% methanolicKOH solution or pure 20% methanolic KOH solution only.

[0020] Synthesis of the platinum fragments. 1,2-Diaminoethane,1,3-diaminopropane, trans-1,2-diaminocyclohexane and 2,2′-bipyridinewere commercially available and used as ligands to prepare thecorresponding dichloroplatinum(II) complexes according to literatureprocedures. Ethyl DL-2,3-diaminopropionate dihydrochloride, ethylL-2,4-diaminobutanoate dihydrochloride and diethylmeso-4,5-diaminosuberate dihydrochloride were synthesized according toliterature procedures and used as ligands for the preparation of thecorresponding diiodoplatinum(II) complexes.

[0021] Synthesis of the platinum complexes. For the reaction with theporphyrincarboxylic acids cisplatin had to be activated by conversioninto diammine(diaqua)platinum(II) hydroxide. It was reacted with anequimolar amount of the porphyrin ligand in a mixture of CHCl₃, ethanoland water or, in the case of the water-soluble ligand, in pure water.The resulting diammine(malonato)platinum(II) complexes precipitated. Tothe reaction mixture of the water-soluble complex CH₂Cl₂ was added toremove neutral impurities. The aqueous hphase was evaporated to obtainthe product.

[0022] The diamine(dichloro)platinum(II) fragments were activated byconversion into diamine(dihydroxy)platinum(II) species, which werereacted with an equimolar amount of the respective porphyrin malonicacid in a mixture of CH₂Cl₂, ethanol and water or, in the case of thewater-soluble ligand, in pure water. The complexes precipitated. To thewater-soluble complex CH₂Cl₂ was added to remove neutral impurities,before the aqueous phase was evaporated to obtain the product.

[0023] For the reaction with the porphyrinmalonic acids it was necessaryto activate the diamine(diiodo)platinum(II) complexes by conversion intodiamine(dinitrato) platinum(II) species, which are water-soluble. Inthis form they were reacted with an equimolar amount of the porphyrinligands, in a mixture of CH₂Cl₂, ethanol and water. The water-insolublecomplexes precipitated after concentrating the solutions.

[0024] Hematoporphyrin Platinum Derivatives Type

[0025] Synthesis of the porphyrin ligands and the platinum precursors.Hemin was transferred to protoporphyrin dimethylester, from which allthe subsequent reactions started. First, protoporphyrin dimethylesterwas treated with 30% hydrobromic acid in acetic acid to give the labileMarkownikoff adduct of HBr to the two vinyl double bonds, which wasreacted with different types of alcohols to replace bromide by thecorresponding alkoxides. As alcohols we chose hydrophilic oligo- andpolyethyleneglycol monomethylethers. During the etherification the HBrformed catalyzed the transesterification of the methylesters into theesters of the corresponding alcohols. The etherified hematoporphyrinesters were purified by column chromatography. The carboxylic acids,which were required for coordination to the platinum(II) moieties, wereprepared by hydrolysis of the esters with 20% methanolic KOH solution.

[0026] 1,2-Diaminoethane, 1,3-diaminopropane,trans-1-2-diaminocyclohexane and 2,2′-bi-pyridine were commerciallyavailable and used as ligands to prepare the correspondingdichloroplatinum(II) complexes according to literature procedures. EthylDL-2,3-diaminopropionate dihydrochloride, ethyl L-2,4-diaminobutanoatedihydrochloride and diethyl meso-4,5-diaminosuberate dihydrochloridewere synthesized according to literature procedures and used as ligandsfor the preparation of the corresponding diiodoplatinum(II) complexes.

[0027] Synthesis of the platinum complexes. Reaction of the porphyrincarboxylic acids with cisplatin did not result in the desired complexes.Therefore, cisplatin had to be activated by conversion intodiammine(diaqua)platinum(II) hydroxide, which was reacted with anequimolar amount of the porphyrin ligand in a mixture of ethanol andwater or, in the case of the water-soluble ligands, in pure water. Theresulting diammine(dicarboxylato)platinum(II) complexes precipitated. Tothe reaction mixtures of the water-soluble complexes CH₂Cl₂ was added toremove neutral impurities before the aqueous phase was evaporated toobtain the products.

[0028] The diamine(dichloro)platinum(II) precursors were activated byconversion into diamine(dihydroxy)platinum(II) species, which werereacted with an equimolar amount of the respective porphyrin carboxylicacid in a mixture of ethanol and water or, in the case of thewater-soluble ligands, in pure water. The complexes precipitated. To thewater-soluble complex CH₂Cl₂ was added to remove neutral impurities andthe aqueous phase was evaporated to obtain the product.

[0029] For the reaction with the porphyrincarboxylic acids it isnecessary to activate the diamine(diiodo)platinum(II) complexes byconversion into diamine(dinitrato) platinum(II) species, which arewater-soluble. In this form they were reacted with an equimolar amountof the porphyrin ligand in a mixture of ethanol and water or, in thecase of the water-soluble ligand, in pure water. The water-insolublecomplexes precipitated after concentrating the solution. Thewater-soluble complexes were isolated by chromatography on silica.

[0030] Exemplary Embodiments

[0031] The following examples are intended to explain the invention inmore detail. The inventive compounds are tetraarylporphyrin platinumderivatives, covered by way of example by examples 1 and 2, andhematoporphyrin platinum derivatives, covered by way of example byexamples 3, 4 and 5.

EXAMPLES Example 1

[0032]

[0033]Diammine[2-(4-{10,15,20-tris[4-(1,4,7-trioxaoctyl)phenyl]porphyrin-5-yl}phenoxy)malonato]platinum(II)(No. 21 in FIG. 1)

[0034] The compound2-(4-{10,15,20-Tris[4-(1,4,7-trioxaoctyl)phenyl]porphyrin-5-yl}phenoxy)malonicacid (109 mg, 0.100 mmol) was dissolved in 10 ml of CHCl₃ and 20 ml ofEtOH, combined with 0.100 mmol of the aqueousdiammine(diaqua)platinum(II) hydroxide solution and stirred for 20 h.Yield: 81.0 mg (54.2 μmol, 54%) purple powder, mp 213-214° C.

[0035] Anal. (C₆₂H₆₆N₆O₁₄Pt.10H₂O, 1494,5) C: calcd. 49,83; found.49,19. H, N: calcd. 5,62; found 6.09.

Example 2

[0036]

[0037](+)-trans-1,2-Diaminocyclohexane[2-(4-{10,15,20-tris[4-(1,4,7,10-tetraoxaundecyl)phenyl]porphyrin-5-yl}phenoxy)malonato]platinum(II)(No. 29 in FIG. 1).

[0038] 122 mg (0.100 mmol) Of the compound2-(4-{10,15,20-Tris[4-(1,4,7,10-tetraoxaundecyl)phenyl]porphyrin-5-yl}phenoxy)malonicacid in 10 ml of CH₂Cl₂ and 20 ml of EtOH were reacted with 0.100 mmolof activated (+)-trans-1,2-diaminocyclohexane(dichloro)platinum(II).Yield: 113 mg (73.9 μmol, 74%) purple solid, mp 208° C. Anal.(C₇₄H₈₆N₆O₁₇Pt, 1526.6) C, H, N.

Example 3

[0039]

[0040]Diammine{7,12-bis[1-(1,4,7-trioxaoctyl)ethyl]-3,8,13,17-tetramethylporphyrin-2,18-dipropionato}platinum(II)(No. 21 in FIG. 2).

[0041] The compound7,12-Bis[1-(1,4,7-trioxaoctyl)ethyl]-3,8,13,17-tetramethylporphyrin-2,18-dipropionicacid (80.3 mg, 0.100 mmol) was dissolved in 6 ml EtOH, combined with0.100 mmol of the aqueous diammine(diaqua)platinum(II) hydroxidesolution and stirred for 20 h. Yield: 23.0 mg (22.3 μmol, 22%) darkbrown powder, mp>250° C. Anal. (C₄₄H₆₂N₆O₁₀Pt, 1030.1). C: calcd. 51.30;found. 50.75. H: calcd. 6.07; found. 5.49. N

Example 4

[0042]

[0043](+)-trans-1,2-Diaminocyclohexane{7,12-bis[1-(1,4,7,10,13,16-hexaoxaheptadecyl)ethyl]-3,8,13,17-tetramethylporphyrin-2,18-dipropionato}platinum(II)(No. 38 in FIG. 2).

[0044] The compound7,12-Bis[1,4,7,10,13,16-hexaoxaheptadecyl)ethyl]-3,8,13,17-tetramethylporphyrin-2,18-dipropionicacid (107 mg, 0.100 mmol) in 10 ml of EtOH were reated with 0.100 mmolof activated (+)-trans-1,2-Diaminocyclohexane(dichloro)platinum(II).

[0045] Yield: 25.5 mg (17.2 μmol, 17%) reddish brown powder; mp 245° C.Anal. (C62H94N6O16Pt.6 H2O, 1482,6). C: calcd. 50.23; found. 49.02. H:calcd. 7.21; found. 6.33. N: calcd. 5,67; found. 6.41.

Example 5

[0046]

[0047]2,2′-Bipyridyl{7,12-bis[1-(1,4,7-trioxaoctyl)ethyl]-3,8,13,17-tetramethylporphyrin-2,18-dipropionato}platinum(II)(No. 40a in FIG. 2).

[0048] 42.2 mg (0.100 mmol) of the compound2,2′-Bipyridyl(dichloro)platinum(II) were suspended in 15 ml of H₂O.After 10 min ultrasonic treatment 34.0 mg (0.200 mmol) of AgNO₃ wereadded and the mixture was stirred for 4 h in the dark at roomtemperature. The precipitated AgCl was filtered off and washed withwater. The filtrate containing the activated platinum(II) complex wasevaporated. The residue was dissolved in 5 ml of H₂O and combined with asolution of 80.3 mg (0.100 mmol)7,12-Bis[1-(1,4,7-trioxaoctyl)ethyl]-3,8,13,17-tetramethylporphyrin-2,18-dipropionicacid in 10 ml of EtOH. After stirring for 20 h at 50° C. and cooling toroom temperature the precipitated solid was filtered, washed with waterand EtOH and dried in vacuo.

[0049] Yield: 64.0 mg (55.5 μmol, 55%) dark purple powder, mp>250° C.Anal. (C₅₄H₆₄N₆O₁₀Pt, 1152.2) C, H, N.

[0050] Biological Data.

[0051] Data of the cytotoxic effect was obtained, for instance, on thehuman tumor cell lines TCC-SUP and J82. The effect of the compounds wasinvestigated in the dark and under irradiation with light at awavelength of 600-730 nm. Selected compounds are clearly more activcytotoxically under irradiation. There is a synergism between thecytotoxic effect of the platinum component and the photodynamicprinciple.

[0052] Cell Lines and General Procedures.

[0053] To determine the antiproliferative activity of the new porphyrinligands and the corresponding platinum complexes with different aminenon-leaving groups two bladder cancer cell lines TCC-SUP and J82 wereselected as in vitro models.

[0054] To discriminate between the cytotoxic and phototoxic effects allexperiments were carried out in duplicate. The cells were seeded intomicroplates and the test compounds were added after 48 h. One batch ofthe microplates was kept in the dark until the end of the experiment,whereas the other microplates were irradiated 48 h after addition of thesubstances for 10 min with a light dose of 24 J·cm⁻², before the plateswere reincubated in the dark.

[0055] End-Point Chemosensitivity Assay.

[0056] Hematoporphyrin Platinum Derivatives Type.

[0057] At a dosage of 1 μM, both the dark- and phototoxicity of theporphyrin-platinum conjugates are influenced by the type of thenon-leaving group. The platinum complexes with 2,2′-bipyridyl (40, 41),ethyl DL-2,3-diaminopropionate (42-46), ethyl DL-2,3-diaminobutanoate(47-51), diethyl meso-4,5-diaminosuberate (52-55) ligands were inactiveat a concentration of 1 μM, both in the dark and after irradiation. Thecompounds bearing 1,2-diaminoethane (27-30) and 1,2-diaminopropane(31-34) non-leaving groups were also inactive against TCC-SUP cells. Themost interesting porphyrin-platinum conjugates were those with thediammine (21-26) and the (+)-trans-1,2-diaminocyclohexane (35-39)ligands. Within these series of compounds the water-soluble complexes 26and 39 were most active with T/C_(corr.) of around 30% and 15%,respectively. At 1 μM concentration the reference cisplatin had aT/C_(corr.) value of approximately 2%. At this dosage there was nostatistically significant enhancement of the cytotoxicity by irradiationof the bladder cancer cells.

[0058] An increase in the concentration of complexes 40-55 to 5 μMresulted in no or only marginal augmentation of the dark toxicity (FIG.2). For most of these complexes the phototoxicity is not much higherthan the cytotoxicity observed without irradiation. However, for 42, 45,47, 49, 50 and 53 there is a distinct effect and for 40 and 44 a verystrong effect on the proliferativation of the TCC-SUP cells uponirradiation is observed (FIG. 2). The highest synergism was found forcompound 52 resulting in the lysis of the tumor cells.

[0059] Apart from cisplatin, the highest antitumor activities weremeasured within the series of porphyrin-platinum conjugates bearingdiammine (21-26) and (+)-trans-1,2-diaminocyclohexane (35-39)non-leaving groups. The differences between dark and light-inducedtoxicities were best for the water-soluble porphyrin-platinum complexes26 and 39 with a side chain length of n≅17 in position 7 and 12 of theporphyrin leaving group. All the ethylenediamine and propylenediaminecomplexes 27-34 showed a remarkable light-induced toxicity (FIG. 2).

[0060] Tetraarylporphyrin Platinum Derivatives Type.

[0061] At a dosage of 1 μM and 5 μM, both the dark- and phototoxicity ofthe tetraarylporphyrin-platinum conjugates 21-38 were highly influencedby the type of the non-leaving group the results agreeing with those ofthe hematoporphyrin-platinum complexes discussed above. 23, 29 and 30were the most active tetraarylporphyrin-platinum conjugates withT/C_(corr.) values of around 37%, 57% and 63%, respectively, at 1 μMconcentration. This is analogous to the hematoporphyrin-platinumcomplexes, the most active of which were those with the diammine or the(+)-trans-1,2-diaminocyclohexane non-leaving groups. At 1 μMconcentration there was only a slight enhancement of the cytotoxicity ofthe tetraarylporphyrin-platinum conjugates with the side chain lengthn=2 and n=3 upon irradiation. On the average the light-inducedT/C_(corr.) values were by approximately 20% lower than the dark-onlycytotoxicities (data not shown). An increase in the concentration of thecomplexes to 5 μM enhanced the dark effects and the phototoxicities asshown in FIG. 1. Apart from cisplatin, the highest antitumor activitieswere measured for the tetraarylporphyrin-platinum conjugates bearingdiammine (21-23) and (+)-trans-1,2-diaminocyclohexane (28-30)non-leaving groups. The differences between dark and light-inducedtoxicities were best for the tetraarylporphyrin-platinum complexes 24,27, 32-34, 36 and 38 with a side chain length of n=2 or n=3 (FIG. 1).

1. Porphyrin platinum derivatives of the (a) tetraarylporphyrin platinumderivatives type according to formula I

and according to formula II

and (b) hematoporphyrin platinum derivatives type according to formulaIII

and according to formula IV


2. Compounds as shown in formulas I, II, III and IV according to claim1, having one or more asymmetric carbon atoms, in the form of theirracemates, their pure enantiomers and/or their diastereoisomers or inthe form of mixtures of these enantiomers or diastereoisomers.
 3. Use ofthe compounds as shown in formulas I, II, III and IV according to claims1 and 2 as therapeutic active compounds for the production ofmedicaments for the treatment of cancer.
 4. Use of the compounds asshown in formulas I, II, III and IV according to claims 1 and 2 astherapeutic active compounds for the production of medicaments for thephotodynamic treatment of tumors.
 5. Particularly preferred use of thecompounds as shown in formulas I, II, III and IV according to claims 1,2 and 4 as therapeutic active compounds for the production ofmedicaments for the photodynamic treatment of tumors by irradiating withelectromagnetic radiation having a wavelenght of 600 to 730 nm. 6.Medicaments comprising one or more compounds according to claims 1 and 2in addition to customary physiologically tolerable carriers and/ordiluents or auxiliaries.
 7. Process for the production of a medicamentaccording to claim 6, characterized in that one or more compoundsaccording to claims 1 and 2 are processed to give pharmaceuticalpreparations or brought into a therapeutically administrable form usinga customary pharmaceutical carriers and/or diluents or otherauxiliaries.
 8. Use of compounds of the formulas I, II, III or IVaccording to claims 1 and 2 and/or pharmaceutical preparations acordingto claims 6 and 7 on their own or in combination with one another or incombination with carriers and/or diluents or other auxiliaries.